Project Summary To accelerate the understanding of craniofacial developmental biology and dental disease, the FaceBase Consortium and others have generated a great variety of genomic data in mice. Among them, mRNA, microRNA (miRNA), and enhancer data covering embryonic days (E) 10.5 to E14.5 are mostly available, and they are related to development of the lip (fusion of the maxillary and nasal processes/prominences during craniofacial development). The original studies have mainly focused on a specific data type (e.g. mRNA or miRNA). Transcription factors (TFs), enhancers, and noncoding RNAs, including both miRNAs and long noncoding RNAs (lncRNAs), are major gene regulators at transcriptional and post-transcriptional levels. Considering that embryonic development involves dynamic and systematic changes in the cellular systems, in this proposal, we hypothesize that a combinatory investigation of transcriptional (e.g. TF, enhancer) and post- transcriptional (e.g. miRNA, lncRNA) regulation will have better power to unveil crucial regulatory mechanisms during lip development, and its disruption would cause lip-developmental defects. To test this hypothesis, in Specific Aim 1, we will investigate TF-miRNA co-regulation of genes during lip development at E10.5-E14.5. Building on our strong preliminary data collection, evaluation, and regulatory annotation work, we will first build TF-miRNA co-regulation networks using the FaceBase and other available data and then identify critical TFs, miRNAs, and common regulation modules in a temporal fashion (e.g. E11.5 vs. E10.5, E12.5 vs. E11.5, E13.5 vs. E12.5, and E14.5 vs E13.5). In Specific Aim 2, we will investigate TF-lncRNA co-regulation during lip development at E10.5-E14.5, including TF-lncRNA co-regulation in proximal and distal maxillary processes. We will first identify TF and gene pairs (TF-gene), TF-lncRNA pairs, and lncRNA-gene pairs between two conditions (distal vs. proximal, embryonic day d2 vs. d1), and then form TF-lncRNA-gene regulatory networks between the two conditions. We will further integrate enhancer regulation into such regulatory networks. The expected outcome is to understand how co-regulation acts in proximal and distal lip development. In Aim 3, we will experimentally validate the top TF-miRNA and TF-lncRNA co-regulation modules in mouse lip development. We will validate five critical miRNAs and their targets within the top miRNA-TF regulatory modules, as well as five lncRNAs within the top TF-lncRNA regulatory modules in primary cells from the mouse embryonic lip region. In addition, we will test functional significance of these miRNAs and lncRNAs in cell proliferation/survival in primary cells. This proposal will be the first combinatory investigation of transcriptional and post-transcriptional regulatory modules involving mouse embryonic lip development. The expected findings will enhance our understanding of the regulatory systems in lip development and such knowledge will be helpful for better understanding of lip-related disease and development of potential therapeutic strategies.